I've performed some ISH on embryo section for NT3, BDNF, NT4 and IGF, but the staining I've obtined is weak. I tried several changes in the protocol, (like using or not protK, differents incubation temperature, probes concentration etc), but even so, the staining is not strong, in other side, I know that's specific because I don't have any staining using sense probes. I'm looking for labs who does it and could send us some plasmids to obtain new probes, but I didn't have find it... Does someone know it or if and how you could obtain new Plasmid DNAs containing the sequence for these NF?
I really appreciate your help, Monara
I don't work on neurotrophins, but instead of doing ISH you can do smFISH . High specificity and better signal.
Hi Monara. I have never done ISH against neurotrophins, so i cannot help you with this. However, if you should decide on doing IHC against the neurotrophins instead i can recommend checking for antibodies at www.pAbmAbs.com. I have tested several antibodies against neurotrophins and rated them on www.pAbmAbs.com and so have others scientist from around the world, so this will probably save you time and money :)
I know this was not what you were asking for, but i hope this helps anyway.
Kind regards,
i really appreciate this site, Simon... I'll let some comments there for the Abs that I've used. I did already IHF and I would like to confirm my results with IHS... even so, thanks.
I work with mice embryos, E10 to E15. I detect with anti-DIG antibodies and visualize with NBT+BCIP.
How long do you develop staining (NBT+BCIP)?
I have to many questions... ;) better show the protocol according to which you do hybridization.
If you are not satisfied with the held probes, you can clone the fragments of sequences of NF into a plasmid and make your own probes.
Hello Piotr,
the protocol is good because for the + control, I have good staining. I think the staining I have obtained for NF is not good because of the probes (too old, I'm not sure about the sequences and if these sequences are expressed in embryo tissue etc). I think the best way to make sure of the staining I have had is performing ISH with new probes. The probes we have, we made them from plasmid gave to us long time ago.
Then, I'm looking for companies that make RNA probes, but it has not been easy to find one that make RNA probes from 400pb, usually, they do oligonucleotides. Do you know which differences between ISH from oligo and long probes (around 800pb)?
I reall appreciate your help.
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